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Title: Method for high-volume sequencing of nucleic acids: random and directed priming with libraries of oligonucleotides

Abstract

Random and directed priming methods for determining nucleotide sequences by enzymatic sequencing techniques, using libraries of primers of lengths 8, 9 or 10 bases, are disclosed. These methods permit direct sequencing of nucleic acids as large as 45,000 base pairs or larger without the necessity for subcloning. Individual primers are used repeatedly to prime sequence reactions in many different nucleic acid molecules. Libraries containing as few as 10,000 octamers, 14,200 nonamers, or 44,000 decamers would have the capacity to determine the sequence of almost any cosmid DNA. Random priming with a fixed set of primers from a smaller library can also be used to initiate the sequencing of individual nucleic acid molecules, with the sequence being completed by directed priming with primers from the library. In contrast to random cloning techniques, a combined random and directed priming strategy is far more efficient.

Inventors:
 [1]
  1. Stony Brook, NY
Issue Date:
Research Org.:
Associated Universities, Inc., Upton, NY (United States)
OSTI Identifier:
869839
Patent Number(s):
5407799
Application Number:
08/135,317
Assignee:
Associated Universities, Inc. (Washington, DC)
Patent Classifications (CPCs):
C - CHEMISTRY C12 - BIOCHEMISTRY C12Q - MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS
DOE Contract Number:  
AC02-76CH00016
Resource Type:
Patent
Country of Publication:
United States
Language:
English
Subject:
method; high-volume; sequencing; nucleic; acids; random; directed; priming; libraries; oligonucleotides; methods; determining; nucleotide; sequences; enzymatic; techniques; primers; lengths; 10; bases; disclosed; permit; direct; 45; 000; base; pairs; larger; necessity; subcloning; individual; repeatedly; prime; sequence; reactions; acid; molecules; containing; octamers; 14; 200; nonamers; 44; decamers; capacity; determine; cosmid; dna; fixed; set; library; initiate; completed; contrast; cloning; combined; strategy; efficient; acid molecule; nucleotide sequences; nucleotide sequence; acid molecules; nucleic acid; nucleic acids; permit direct; directed priming; base pairs; base pair; /435/

Citation Formats

Studier, F William. Method for high-volume sequencing of nucleic acids: random and directed priming with libraries of oligonucleotides. United States: N. p., 1995. Web.
Studier, F William. Method for high-volume sequencing of nucleic acids: random and directed priming with libraries of oligonucleotides. United States.
Studier, F William. Tue . "Method for high-volume sequencing of nucleic acids: random and directed priming with libraries of oligonucleotides". United States. https://www.osti.gov/servlets/purl/869839.
@article{osti_869839,
title = {Method for high-volume sequencing of nucleic acids: random and directed priming with libraries of oligonucleotides},
author = {Studier, F William},
abstractNote = {Random and directed priming methods for determining nucleotide sequences by enzymatic sequencing techniques, using libraries of primers of lengths 8, 9 or 10 bases, are disclosed. These methods permit direct sequencing of nucleic acids as large as 45,000 base pairs or larger without the necessity for subcloning. Individual primers are used repeatedly to prime sequence reactions in many different nucleic acid molecules. Libraries containing as few as 10,000 octamers, 14,200 nonamers, or 44,000 decamers would have the capacity to determine the sequence of almost any cosmid DNA. Random priming with a fixed set of primers from a smaller library can also be used to initiate the sequencing of individual nucleic acid molecules, with the sequence being completed by directed priming with primers from the library. In contrast to random cloning techniques, a combined random and directed priming strategy is far more efficient.},
doi = {},
journal = {},
number = ,
volume = ,
place = {United States},
year = {1995},
month = {4}
}

Works referenced in this record:

Carving up the human genome
journal, December 1988


A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity
journal, July 1983


The use of a random priming procedure to generate cDNA libraries of infectious bronchitis virus, a large RNA virus
journal, July 1985


Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing
journal, October 1980